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Long-term stabilization of DNA at room temperature using a one-step microwave assisted process.

ABSTRACT: Long-term stabilization of DNA is needed for forensic, clinical, in-field operations and numerous other applications. Although freezing (<-20 °C) and dry storage are currently the preferential methods for long-term storage, a noticeable pre-analytical degradation of DNA over time, upfront capital investment and recurring costs have demonstrated a need for an alternative long-term room-temperature preservation method. Herein, we report a novel, fast (~5 min) silica sol-gel preparation method using a standard microwave-initiated polymerization reaction amenable to stabilization of DNA. The method involves use of one chemical, tetramethoxy silane (TMOS) and eliminates the use of alcohol as co-solvent and catalysts such as acids. In addition, the process involves minimal technical expertise, thus making it an ideal choice for resource-challenged countries and in-field applications. The sol-gel is capable to store and stabilize Escherichia coli DNA in ambient conditions for 210 days. DNA recovered from the sol-gel showed no significant nucleolytic and/or oxidative degradation, outperforming conventional storage conditions at -20 °C, and reported state-of-the-art technology.

Supplementary information

The online version contains supplementary material available at 10.1007/s42247-021-00208-3.

SUBMITTER: Narvaez Villarrubia CW

PROVIDER: S-EPMC7986179 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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Long-term stabilization of DNA at room temperature using a one-step microwave assisted process.

Narvaez Villarrubia Claudia W CW Tumas Keyla C KC Chauhan Rajat R MacDonald Thomas T Dattelbaum Andrew M AM Omberg Kristin K Gupta Gautam G

Emergent materials 20210323 2

Long-term stabilization of DNA is needed for forensic, clinical, in-field operations and numerous other applications. Although freezing (<-20 °C) and dry storage are currently the preferential methods for long-term storage, a noticeable pre-analytical degradation of DNA over time, upfront capital investment and recurring costs have demonstrated a need for an alternative long-term room-temperature preservation method. Herein, we report a novel, fast (~5 min) silica sol-gel preparation method usin ...[more]

PMID: 33778372

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Project description:BackgroundThere is an urgent need to measure phosphorylated cell signaling proteins in cancer tissue for the individualization of molecular targeted kinase inhibitor therapy. However, phosphoproteins fluctuate rapidly following tissue procurement. Snap-freezing preserves phosphoproteins, but is unavailable in most clinics and compromises diagnostic morphology. Formalin fixation preserves tissue histomorphology, but penetrates tissue slowly, and is unsuitable for stabilizing phosphoproteins. We originated and evaluated a novel one-step biomarker and histology preservative (BHP) chemistry that stabilizes signaling protein phosphorylation and retains formalin-like tissue histomorphology with equivalent immunohistochemistry in a single paraffin block.ResultsTotal protein yield extracted from BHP-fixed, routine paraffin-embedded mouse liver was 100% compared to snap-frozen tissue. The abundance of 14 phosphorylated proteins was found to be stable over extended fixation times in BHP fixed paraffin embedded human colon mucosa. Compared to matched snap-frozen tissue, 8 phosphoproteins were equally preserved in mouse liver, while AMPKβ1 Ser108 was slightly elevated after BHP fixation. More than 25 tissues from mouse, cat and human specimens were evaluated for preservation of histomorphology. Selected tissues were evaluated in a multi-site, independent pathology review. Tissue fixed with BHP showed equivalent preservation of cytoplasmic and membrane cytomorphology, with significantly better nuclear chromatin preservation by BHP compared to formalin. Immunohistochemical staining of 13 non-phosphorylated proteins, including estrogen receptor alpha, progesterone receptor, Ki-67 and Her2, was equal to or stronger in BHP compared to formalin. BHP demonstrated significantly improved immunohistochemical detection of phosphorylated proteins ERK Thr202/Tyr204, GSK3-α/β Ser21/Ser9, p38-MAPK Thr180/Tyr182, eIF4G Ser1108 and Acetyl-CoA Carboxylase Ser79.ConclusionIn a single paraffin block BHP preserved the phosphorylation state of several signaling proteins at a level comparable to snap-freezing, while maintaining the full diagnostic immunohistochemical and histomorphologic detail of formalin fixation. This new tissue fixative has the potential to greatly facilitate personalized medicine, biobanking, and phospho-proteomic research.

Dataset Information

Long-term stabilization of DNA at room temperature using a one-step microwave assisted process.

Supplementary information

Publications

Long-term stabilization of DNA at room temperature using a one-step microwave assisted process.

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